Catherine Felgines

Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice ☆

Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoE⁻/⁻) mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo E⁻/⁻ mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell-cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.

Involvement of glutamine, arginine, and polyamines in the action of ornithine alpha-ketoglutarate on macrophage functions in stressed rats.

The ability of ornithine α‐ketoglutarate (OKG) to enhance macrophage cytotoxicity in stress situations has been described, but the mechanisms involved remain unclear. It is known that OKG administration generates glutamine (GLN), arginine (ARG), and polyamines. This study will (1) evaluate the effect of OKG on tumor necrosis factor α (TNF‐α) secretion and nitric oxide (NO) production in macrophages from glucocorticoid (DEX)‐treated rats, and determine whether these effects can be reproduced by GLN or ARG supplementations, and (2) use in vivo metabolic inhibitors methionine sulfoximine (inhibitor of GLN synthetase), S‐methylthiourea (inhibitor of inducible nitric oxide synthase), and difluoromethylornithine (inhibitor of ornithine decarboxylase) to assess the roles of GLN, ARG, and polyamines in OKG action. Controls a mixture of nonessential amino acids (NEAA). GLN, ARG, and OKG all restored TNF‐α secretion by macrophages of glucocorticoid‐treated rats. The same results were obtained with GLN and ARG supplementation. However, the use of inhibitors clearly showed that OKG does not modulate TNF‐α secretion by GLN, ARG, or polyamine pathways. We also observed that OKG enhanced NO release by stimulated macrophages (DEX‐OKG, 1.77 ± 0.64 vs. DEX‐NEAA, 0.29 ± 0.29 nmol/106 cells, P < 0.05). Using inhibitors, it appears that this action of OKG is probably mediated via polyamine synthesis and GLN. However, an oral administration of an equimolar amount of GLN failed to reproduce the OKG‐mediated effect, possibly because OKG generates more GLN in the systemic circulation than GLN itself when these substances are given orally. Our results underline the complexity of the mechanism of action of OKG, which can differ according to the functions of even a single cell type. J. Leukoc. Biol. 67: 834–840; 2000.

Strawberry pelargonidin glycosides are excreted in urine as intact glycosides and glucuronidated pelargonidin derivatives in rats

Anthocyanins are natural dietary pigments with a wide array of biological properties that are possibly involved in the prevention of various diseases. These properties depend on their absorption and metabolism in the body. In the present study we first examined the gastric and intestinal absorption of pelargonidin 3-glucoside (Pg 3-glc) using rat in situ models. A high proportion of Pg 3-glc was rapidly absorbed from both the stomach (23 %) and small intestine (24 %). Its metabolism was further studied by feeding rats during 8 d with a diet enriched in freeze-dried strawberries. Only low amounts of total anthocyanins were recovered in 24 h urine (0.163 (SEM 0.013) % of ingested anthocyanins; n 8). Strawberry anthocyanins were analysed in urine by HPLC-electrospray ionisation-tandem MS. Similar proportions of intact glycosides (about 53 %) and glucuronidated metabolites (about 47 %) were found. Pg 3-glc was thus glucuronidated to a larger extent than cyanidin 3-glucoside. These results highlight the influence of the aglycone structure on anthocyanin metabolism.

Radiolabelled cyanidin 3-O-glucoside is poorly absorbed in the mouse.

Anthocyanins are natural pigments abundant in various fruits and berries that are involved in the prevention of various chronic diseases. Their low concentrations in plasma and urine are explained in part by their complex chemistry and the formation of still uncharacterised metabolites. The aim of the present study was to follow the distribution of anthocyanins in the body using 14C-labelled cyanidin 3-O-glucoside (Cy3G) fed by gavage to mice. After the administration of 22.2 kBq 14C-Cy3G (0.93 mg), radioactivity was detected in most organs tested over the following 24 h with a peak observed in inner tissues at 3 h. The major fraction of the radioactivity (44.5 %) was found in the faeces collected 24 h after ingestion. At 3 h after oral administration of 141 kBq 14C-Cy3G (4.76 mg), most of the radioactivity (87.9 % of intake) was recovered in the gastrointestinal (GI) tract, especially in the small intestine (50.7 %) and the caecum (23 %). At this time, 3.3 % of the radioactivity was detected in urine. There was minimal accumulation (0.76 %) of radioactivity in tissues outside the GI tract. Distribution of radioactivity varied among organs, with liver, gallbladder and kidneys showing the highest radioactivity. Taken as a whole, these results show that Cy3G is poorly absorbed in the mouse.

Atheroprotective Effects of Bilberry Extracts in Apo E-Deficient Mice

Previous studies have demonstrated that the intake of berry foods was associated with a reduced risk of cardiovascular diseases. The aim of the present study was to evaluate the effects of two bilberry extracts, one rich in anthocyanins extracted from untreated bilberries (BE) and a second one extracted from yeast-fermented bilberries (FBE), on the development of atherosclerosis in apolipoprotein E-deficient mice (apo E(-/-)). Apo E(-/-) mice received for 16 weeks a diet supplemented with 0.02% of either BE or FBE. Atherosclerotic plaque area was measured in the aortic sinus. Supplementation of the diet with both bilberry extracts led to a significant inhibition of plaque development, whereas no effect on oxidative stress parameters or lipid profiles could be observed, suggesting the implication of other mechanisms of action. In addition, a better protection was observed with FBE, suggesting that the fermentation generates new bioactive compounds more effective in attenuating progression of the atherosclerotic lesions.

Protein metabolism in rats during long-term dietary restriction : Influence of aging

BACKGROUND Protein depletion is frequent in the elderly, but the underlying mechanisms are not yet fully understood. In particular, it is unknown whether there is a defect of adaptation to a restriction of food intake in the elderly. This study was performed to compare the effects of 6-week dietary restriction (DR) on protein metabolism in both adult and aged rats. METHODS Adult (3-month-old) and aged (22-month-old) rats were acclimatized for 2 weeks and then fed a standard diet for 6 weeks, either ad libitum (control adult [C(Adult)] and aged [C(Aged)] rats) or with only 50% of the average intake of the second week of acclimatization (restricted adult [R(Adult)] and aged [R(Aged)] rats). Protein metabolism, in terms of tissue protein content, nitrogen balance, and 3-methylhistidine (3-MH) urinary excretion, was evaluated. RESULTS C(Adult) rats gained 30.4% of initial weight, whereas the body weight (BW) of C(Aged) rats was maintained. DR induced a rapid decrease in BW during the first 2 weeks in R(Adult) rats, but afterward BW remained stable. In R(Aged) rats, BW loss was linear during the 6 weeks and significantly higher than for R(Adult) rats (p<.01). In both restricted groups, muscle protein content was moderately affected by DR, whereas DR induced a marked decrease in visceral protein content. Nitrogen balance was decreased by DR but stayed positive in R(Adult) rats, whereas it became null in R(Aged) rats. CONCLUSIONS In terms of protein metabolism, aged rats adapted less efficiently than adult rats to a long-term dietary restriction.

Tissue distribution of anthocyanins in rats fed a blackberry anthocyanin-enriched diet.

Anthocyanins are natural dietary pigments that could be involved in various health effects. The aim of this study was to investigate the distribution of anthocyanins to various organs (bladder, prostate, testes, heart and adipose tissue) in rats fed with a blackberry anthocyanin-enriched diet for 12 days. Identification and quantification of anthocyanins were carried out by HPLC-DAD. The urinary excretion of total anthocyanins (native anthocyanins and their metabolites) was low (0.20 +/- 0.03%, n = 8). Proportions of anthocyanin derivatives (methylated anthocyanins and glucurono-conjugated derivatives) differed according to the organ considered. The bladder contained the highest levels of anthocyanins followed by the prostate. Prostate, testes and heart contained native cyanidin 3-glucoside and a small proportion of cyanidin monoglucuronide. Cyanidin 3-glucoside and methylated derivatives were present in adipose tissue. Thus, anthocyanin feeding in rats resulted in a wide distribution of anthocyanin derivatives to several organs. Identification of target tissues of anthocyanins may then help to understand the mechanisms of action of anthocyanins in vivo.

Effects of dietary fermentable fiber on fatty acid synthesis and triglyceride secretion in rats fed fructose-based diet : studies with sugar-beet fiber

Abstract In an attempt to elucidate the role of the dietary fermentable fiber in reduction of hyperlipidemia, we substituted 30% wheat starch with 30% sugar-beet fiber in rats fed a fructose-based (41% fructose), low-fat (2% corn oil) diet. Male Wistar rats ate the test diets for 3 weeks. Feeding the sugar-beet fiber (SBF) diet resulted in a significant enlargement of the cecum; it also increased the concentration of volatile fatty acids compared with rats fed a fiber-free (FF) diet. Feeding SBFdecreased plasma triglyceride and cholesterol concentrations in the postprandial as well as the postabsorptive period. In the liver, triglyceride levels were depressed in concert with the decreased liver lipogenesis and the post-Triton triglyceride secretion. Liver cholesterol levels were unaffected by SBF diet feeding. SBF-fed animals were markedly less fat compared with fiber-free-diet-fed rats. Adipose tissue lipogenesis was depressed in the postprandial period in SBF-fed animals. In short, this study suggests that substitution of easily digested carbohydrates by certain fermentable fibers may play an interesting role in the reduction of hyperlipidemia and obesity.

Influence of glucose on cyanidin 3-glucoside absorption in rats.

Anthocyanins are natural dietary pigments that could be involved in various health effects. However their mechanisms of absorption are still not fully understood. The aim of this study was to evaluate the influence of glucose on anthocyanin absorption in rats. We first studied anthocyanin bioavailability in rats that received by gastric intubation approximately 53 micromol cyanidin 3-glucoside (Cy 3-glc) equivalents from a red orange extract with or without 2.51 mmol glucose. Neither 24-h urinary anthocyanin excretion nor plasma anthocyanin concentration was significantly affected by simultaneous ingestion of glucose. The influence of glucose (12, 42 or 72 mM) on intestinal absorption of Cy 3-glc (pure or from a red orange extract; approximately 12.3 microM) was further studied using an in situ intestinal perfusion model. Absorption of pure Cy 3-glc from the intestinal lumen was not significantly affected by the amount of glucose. However, intestinal absorption of Cy 3-glc from the red orange extract (6.49 +/- 1.44%, n = 6) was significantly less than that of pure Cy 3-glc (17.5 +/- 1.3%, n = 7) (p < 0.01) suggesting that the red orange extract contained other components that were able to interfere with Cy 3-glc intestinal absorption. This study has thus shown that glucose did not interfere with anthocyanin glucoside absorption.

Effect of simvastatin treatment on plasma apolipoproteins and hepatic apolipoprotein mRNA levels in the genetically hypercholesterolemic rat (RICO)

The effects of long-term treatment with simvastatin on plasma lipoproteins, plasma apolipoproteins, and on hepatic apolipoprotein gene expression were evaluated in genetically hypercholesterolemic (RICO) rats. Simvastatin administration caused a decrease in plasma triglyceride and phospholipid concentrations. Plasma cholesterol concentration was not changed by simvastatin, but cholesterol distribution among plasma lipoproteins was altered. Plasma apo B, apo A-I, and apo A-IV concentrations were lowered by simvastatin treatment whereas plasma apo E concentration was not affected by this drug. In the liver, simvastatin treatment induced a significant decrease of apo E mRNA level but had no effect on apo B, apo A-I, and apo A-IV mRNA abundances. It appears that simvastatin may modify plasma apolipoprotein concentrations by influencing their hepatic synthesis at both pre- and posttranscriptional levels.